Phoenix 7

Plasma samples were thawed on ice. Blank mouse plasma was used for standard curves. Plasma aliquots (100 ul) were spiked with hesperetin (10 ul, 5000 ng/ml) as an internal standard and mixed by vortexing. Drugs were extracted with simple protein precipitation using cold acetonitrile, and the mixture was centrifuged at 13000 × g for 10 min. The supernatant was collected and dried under nitrogen. Dried samples were reconstituted with 200 ul 1% formic acid. Samples were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) system consisting of Thermo Accela UHPLC pump, Thermo PAL autosampler, and Thermo TSQ Discovery triple quadruple mass spectrometer (Thermo, Waltham, MA, USA). Chromatographic separation was done using an Agilent Zobrax Extend C-18 column. Linear gradient elution was used at a flow rate of 400 ul/min, with a mobile phase of 0.5% acetonitrile and 95% acetonitrile, modified with 0.2% formic acid. Non-compartmental analysis of the pharmacokinetic parameters as well as compartmental model fitting of the data was carried using Phoenix^® 7.0 (Certara, New Jersey, USA).